This invention relates generally to an apparatus and method for an improved nanocalorimeter, and more specifically, to a system and method for an improved nanocalorimeter for measuring the heat released or absorbed during chemical reactions.
Calorimetry is used to measure enthalpic changes, including enthalpic changes arising from reactions, phase changes, changes in molecular conformation, temperature variations, and other variations of interest that may occur for a particular specimen. By measuring enthalpic changes over a series of conditions, other thermodynamic variables may be deduced. For example, measurements of enthalpy as a function of temperature reveal the heat capacity of a specimen, and titrations of reacting components can be used to deduce the binding constant and effective stoichiometry for a reaction. Calorimetry measurements are useful in a broad variety of applications, including, for example, pharmaceuticals (drug discovery, decomposition reactions, crystallization measurements), biology (cell metabolism, drug interactions, fermentation, photosynthesis), catalysts (biological, organic, or inorganic), electrochemical reactions (such as in batteries or fuel cells), and polymer synthesis and characterization, to name a few. In general, calorimetry measurements can be useful in the discovery and development of new chemicals and materials of many types, as well as in the monitoring of chemical processes. Standard calorimeters require relatively large samples (typically about 0.5 ml to 10 liters) and usually measure one sample at a time. As such, these systems cannot be used to measure very small samples, as might be desired for precious or highly reactive materials. Furthermore, standard calorimeters cannot be used effectively to monitor a large number of reactions of small sample size in parallel, as is required in order to perform studies using combinatorial chemistry techniques.
In recent years, researchers and companies have turned to combinatorial methods and techniques for discovering and developing new compounds, materials, and chemistries. For example, pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. As another example, Symyx Technologies® is applying combinatorial techniques to materials discovery in the life sciences, chemical, and electronics industries. Consequently, there is a need for tools that can measure reactions and interactions of large numbers of small samples in parallel, consistent with the needs of combinatorial discovery techniques. Preferably, users desire that these tools enable inexpensive measurements and minimize contamination and cross-contamination problems.
In some cases, the sample to be studied is precious, and it might not be acceptable to use the relatively large amount of material required by a standard microcalorimeter to perform only one measurement. For example, one may desire to study a natural extract or synthesized compound for biological interactions, but in some cases the available amount of material at concentrations large enough for calorimetry might be no more than a few milliliters. Performing a measurement in standard microcalorimeters, such as those sold, for example, by MicroCal® Inc. (model VP-ITC) or Calorimetry Sciences Corporation® (model CSC-4500), requires about 1 ml of sample, which means that one would possibly be faced with using a majority or all of the precious material for one or a small series of measurements. Tools that enable calorimetric measurements with much smaller sample sizes would be helpful in overcoming this limitation.
One of the most popular uses of combinatorial techniques to date has been in pharmaceutical research. Pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. A combinatorial library is a collection of chemical compounds which have been generated, by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” as reagents. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixing of chemical building blocks.
Once a library has been constructed, it must be screened to identify compounds, which possess some kind of biological or pharmacological activity. For example, screening can be done with a specific biological compound, often referred to as a target that participates in a known biological pathway or is involved in some regulation function. The library compounds that are found to react with the targets are candidates for affecting the biological activity of the target, and hence a candidate for a therapeutic agent.
Through the years, the pharmaceutical industry has increasingly relied on high throughput screening (HTS) of libraries of chemical compounds to find drug candidates. HTS describes a method where many discrete compounds are tested in parallel so that large numbers of test compounds are screened for biological activity simultaneously or nearly simultaneously. Currently, the most widely established techniques utilize 96-well microtitre plates. In this format, 96 independent tests are performed simultaneously on a single 8 cm×12 cm plastic plate that contains 96 reaction wells. These wells typically require assay volumes that range from 50 to 500 μl. In addition to the plates, many instruments, materials, pipettors, robotics, plate washers and plate readers are commercially available to fit the 96-well format to a wide range of homogeneous and heterogeneous assays. To achieve faster testing, the industry is evolving to plates that contain 384 and 1536 wells.
A variety of measurement approaches has been used to screen combinatorial libraries for lead compounds, one of which is the inhibitor assay. In the inhibitor assay, a marker ligand, often the natural ligand in a biological pathway, is identified that will bind well with the target protein molecule. The assay requires the chemical attachment of a fluorescent molecule to this marker ligand such that the fluorescent molecule does not affect the manner in which the marker ligand reacts with the target protein. To operate an inhibitor assay, the target protein is exposed to the test ligands in microtitre wells. After a time necessary for reaction of the test ligand to the target protein, the marker ligand is applied. After a time for reaction with the marker ligand, the wells are rinsed such that non-reacted marker ligand is washed away. In wells where the target protein and the test ligand have reacted, the test ligand blocks the active site of the target protein so the marker ligand cannot react and is washed away, while in cells where the target protein and test ligand have not reacted, the marker ligand reacts with the target protein and is not washed away. By investigating the wells for the presence of fluorescence after the washing, reactions of test ligands and target proteins can be determined as having occurred in wells where no fluorescence is observable.
However, the inhibitor assay requires time and expense to develop the assay. The principal components that need development are discovering a marker ligand and attaching a fluorophore to the marker in a manner that does not affect its reaction with the target protein. Attaching the fluorescent marker can often take 3 months of development or more and cost $250 k or more once the marker ligand is identified. An assay method that avoids such assay development, such as measuring the heat of the reaction with calorimetry, would eliminate this cost and time delay in the discovery process.
Calorimetry measurements are commonly utilized in biophysical and biochemical studies to determine energy changes as indications of biochemical reactions in a media. Prior techniques for measurements include using electrodes, thermopiles, optical techniques, and microcalorimeters for measurements within a sampled media. There is a great interest in developing ultra-miniature microcalorimeter devices that require very small volumes of sampled media for accurate detection and measuring of biochemical reactions on, or in close proximity to, the microcalorimeter and which can be applied in a manner to quickly measure large numbers of reactions such that it can be as efficient as assays such as inhibitor assays which can be used in HTS to screen perhaps 100,000 test ligands a day.
The following disclosures may be relevant and/or helpful in providing an understanding of some aspect of the present invention:
In Plotnikov et al., U.S. Pat. No. 5,967,659 (“Ultrasensitive Differential Microcalorimeter with User-selected Gain Setting”), a differential calorimeter is disclosed that includes sample and reference cells, a thermal shield surrounding the cells, heating devices thermally coupled to the thermal shield and the cells, a temperature monitoring system, and a control system. The temperature monitoring system monitors the temperature of the shield, cell temperatures, and temperature differentials between the cells and the shield. The control system generates output signals for control of the heating devices, with a gain setting and scan rate selected by means of a user interface. Output control signals are functions of input temperature signals and the user-selected gain setting, as well as functions of input temperature signals and the user-selected scan rate using a mapping function stored in memory.
In Cavicchi et al., U.S. Pat. No. 6,079,873 (“Micron-scale Differential Scanning Calorimeter on a Chip”), a differential scanning microcalorimeter produced on a silicon chip enables microscopic scanning calorimetry measurements of small samples and thin films. The chip, fabricated using standard CMOS processes, includes a reference zone and a sample zone. The reference and sample zones may be at opposite ends of a suspended platform or may reside on separate platforms. Each zone is heated with an integrated polysilicon heater. A thermopile consisting of a succession of thermocouple junctions generates a voltage representing the temperature difference between the reference and sample zones.
In Thundat et al., U.S. Pat. No. 6,096,559 (“Micromechanical Calorimetric Sensor”), a calorimeter sensor apparatus utilizes microcantilevered spring elements for detecting thermal changes within a sample containing biomolecules which undergo chemical and biochemical reactions. The spring element includes a bimaterial layer of chemicals on a coated region on at least one surface of the microcantilever. The chemicals generate a differential thermal stress across the surface upon reaction of the chemicals with an analyte or biomolecules within the sample due to the heat of chemical reactions in the sample placed on the coated region. The thermal stress across the spring element surface creates mechanical bending of the microcantilever. The spring element has a low thermal mass to allow detection and measuring of heat transfers associated with chemical and biochemical reactions within a sample place on or near the coated region. Deflections of the cantilever are detected by a variety of detection techniques.
In Lieberman, U.S. Pat. No. 6,193,413 (“System and Method for an Improved Calorimeter for Determining Thermodynamic Properties of Chemical and Biological Reactions”) a microcalorimeter includes a thin amorphous membrane anchored to a frame within an environmental chamber. Thermometers and heaters are placed on one side of a thermal conduction layer mounted on the central portion of the membrane. Samples are placed on two such membranes; each sample is heated and its individual heat capacity determined. The samples are then mixed by sandwiching the two microcalorimeters together to cause a binding reaction to occur. The amount of heat liberated during the reaction is measured to determine the enthalpy of binding.